Finding a Cure: Which HIV vaccine would you choose?

1. Finding a Cure: Which HIV vaccine would you choose? Ramil Sapinoro Life Sciences Learning Center University of Rochester Medical Center

2. AIDSVax Inc. Department of Vaccine Research 1979 Cure Drive Rochester, NY 14620 Dear Research Technician, We are excited to collaborate with your organization in developing a AIDS vaccine. I am sending you 4 possible DNA vaccines that you could use in your vaccine trials. I’m not sure which combinations of the three HIV genes are in each one. You will have to check this. Along with the DNA, I have included some data collected from preliminary animal studies. We injected animals with each of the three HIV genes alone: the gag gene codes for HIV structural proteins; the env gene codes for the virus envelope proteins; and the pol gene codes for viral enzymes. We used mice as an experimental animal. Sincerely, Clarke Kent, Ph.D Director Center for Vaccine Research University of Rochester

3. APC How does the Immune System fight invaders? Antigen MHC Antigen Presenting Cells (ex: macrophages) Capture invaders (pathogens), destroy them, and display parts of them (antigens) on proteins called MHC’s Virus

4. T APC How does the Immune System fight invaders? • Helper T Cells • See antigens presented by APC’s and does two things: • Turn on Cytotoxic T Cell – these T Cells can go on to kill other cells that present the antigen (infected cells as well as macrophages) • Activate B Cells

5. T Cytotoxic T Cell B T APC B Cell How does the Immune System fight invaders?

6. B B B B T How does the Immune System fight invaders? Helper T cell produces signaling proteins that tell B Cells that have antibodies against the antigen to make more B Cells and free floating antibodies Cytotoxic T cell produces signaling proteins that tell infected cells to kill themselves Infected Cell

7. How do vaccines ‘help’ the immune system to fight off viruses?

8. B B What would a Successful Vaccine Do? • Stimulate the production of antibodies • Stimulate the production of Cytotoxic T Cells • Remember the pathogen (Memory cells) • Not cause disease

9. Vaccine ApproachesWhat parts of a pathogen could we use to stimulate the immune response and not cause disease? Live-attenuated Virus Inactivated Virus DNA Protein subunit Synthetic peptide

10. Vaccines Currently Available

11. T APC What is HIV? • HIV = Human Immunodeficiency Virus • Small • Replicates within cells of living hosts • Composed of RNA • Pathogenic: Has the ability to cause disease (AIDS) HIV causes disease because it kills helper T cells

12. HIV Genome and Structure The HIV genome has three main genes… gag gene pol gene env gene env proteins Viral envelope proteins gag proteins Capsid Matrix pol proteins Viral enzymes

13. Four Potential Vaccines V1 V2 V3 V4 gag gene pol gene env gene Which of these possible pieces of DNA are in each vaccine? How will you find out? pol gene env gene gag gene pol gene gag gene env gene pol gene

14. Which genes are in the vaccines? Cut with Restriction Enzyme ? V1 DNA Vaccine ? V2 DNA Vaccine ? V3 DNA Vaccine ? V4 DNA Vaccine

15. Gel electrophoresis of digested vaccines You have been given vaccines that have been pre-digested with restriction enzyme. You’ll need to run the DNA fragments out on a gel to see what genes are in each vaccine. V1 V2 V3 V4

16. Loading your gel

17. Loading your gel

18. Gel electrophoresis of digested vaccines V3 V1 V2 V4 • Load the gel • Carry the gel to a power source • Fill the gelbox with TAE buffer (fill the box so that the top of the gel is just covered) • Put the lid on the gel • Run the gel for about 10 minutes at 120 volts

19. Which genes are in the vaccines? gag gene pol gene env gene Restriction Enzyme gag gene Well pol gene env gene Gel electrophoresis

20. Which genes are in the vaccines? gag gene pol gene Restriction Enzyme gag gene Well pol gene Gel electrophoresis

21. Which genes are in the vaccines? pol gene env gene Restriction Enzyme Well pol gene env gene Gel electrophoresis

22. Which genes are in the vaccines? gag gene Restriction Enzyme gag gene Well Gel electrophoresis

23. Which genes are in the vaccines? pol gene Restriction Enzyme Well pol gene Gel electrophoresis

24. Which genes are in the vaccines? env gene Restriction Enzyme Well env gene Gel electrophoresis

25. Which one of these would you use in a human trial? gag gene pol gene env gene pol gene env gene gag gene pol gene gag gene env gene pol gene Which ones do you KNOW you would NOT want to use? Of the others, how will you decide which ones to use in human trials?

26. B B Vaccine study in an animal model Amount of Antibodies DNA injected into mice Which one(s) would you use to make a vaccine?

27. B B Vaccine study in an animal model Amount of cytotoxic T-cell Activity DNA injected into mice Which one(s) would you use to make a vaccine?

28. More about HIV/AIDS…

29. CD4 Helper T Cell HIV Lifecycle

30. Global HIV infections, Dec 2006

31. HIV Treatment: Anti-virals

32. Reverse Transcription Inhibitors X Protease Blockers X

33. What is in the vaccines? Which ones would you use in human trials? Which genes are in each of the four potential vaccines? Which of the potential vaccines would you NOT use? What other information might you need to make a decision on which one to use? V2 V3 V4 V1

34. Clinical Trials: Testing in Humans

35. Phase Objective Number of Volunteers Safety and dose 20–50 / trial I Additional safety and immunogenicity II 100’s / trial Efficacy and protection 1000’s / trial III

36. Phase Objective Number of Volunteers Safety and dose 20–50 / trial I Additional safety and immunogenicity II 100’s / trial Efficacy and protection 1000’s / trial III V2 and V4

37. You will be testing the serum of a group of people who have either been injected with V2, V4 or a placebo to see if they respond to the vaccine by making antibodies against the appropriate proteins

38. Each volunteer is injected with one vaccine. After a time, serum is collected from the volunteer, and analyzed for the presence of antibodies using an ELISA assay… V2 Serum

39. Obtain three absorbent squares and label “G” (gag) “P” (pol) and “E” (env)…

40. Spot the gag, pol and env proteins on their appropriate spots… Gag Pol Env

41. Place serum on each of the squares – the serum contains antibodies… Gag Pol Env

42. If the volunteer has antibodies against the HIV proteins, those antibodies will bind to the proteins on the paper. Excess antibodies will wash off… Gag Pol Env

43. Add labeled “Secondary Antibody” This secondary antibody binds to all human antibodies, and has a colored tag… Gag Pol Env

44. Upon the addition of “developing solution,” the colored tag will become visible… Gag Pol Env

45. You will each be given one serum sample from one volunteer – follow the instructions in your handout to determine if the volunteer made the appropriate antibodies…

46. Which vaccine worked the best in the trial?

47. APC APC Antigen MHC allele #1 MHC allele #2 Why do we need to test immunogenicity?Diversity, diversity, diversity… People have different MHC alleles Person 1 Immune response Person 2 No immune response

48. MHC Polymorphism

49. Challenges in HIV Vaccine Research • Viral Genetic Diversity: HIV is not just one specific virus. • Immune Protection: Scientists don’t know what immune responses are needed, or how strong they need to be. • Neutralizing Antibody: Difficult to generate broadly neutralizing antibodies. • Vaccine Testing: Slow process, very expensive

50. …but on the Brightside… • Precedent from other systems: Success against other viral infections • Precedent from animal studies: Long-term control of infection in vaccinated monkeys • Immune control of HIV-1: Infected individuals control infection • Vaccine Trials: In progress